Current testing for Lyme disease is suboptimal because it requires two assay platforms, including one with subjective interpretation of results, and because it has lower sensitivity in the early stage of the illness. Infection with Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted by the bite of an Ixodid tick and results in uniformly low pathogen burdens with only a transient bloodborne phase, so that the sensitivity of direct detection assays of the bacteria in easily accessible tissues such as the blood is low. Diagnostic testing therefore relies on serologic assays to detect antibodies to the bacteria. The CDC currently recommends a two-tier assay with an initial ELISA, and positive or equivocal results must be confirmed by immunoblot. Historically both assays were performed with whole cell sonicate of in vitro cultured bacteria even though it is now known that B. burgdorferi spirochetes alter their protein expression when they move from the tick to the mammalian host and when they are cultured in vitro. More recently an ELISA for the C6 peptide from the VlsE protein has been developed and is now sometimes used instead of the lysate ELISA. The results of this assay, which is based on just one antigen, are still often confirmed by immunoblot that is time consuming, subjective, and expensive. We have developed a new one-step rapid multiplex assay using the Luminex xMAP Technology platform that improves upon both the assay platform of the current tests and the antigens used. We identified a panel of antigens that are expressed by spirochetes within the mammalian host and to which early IgG responses are generated. We have developed a multiplex assay that has a much larger dynamic range than ELISA or immunoblot, and can be completed more rapidly and objectively. The use of multiple but specific antigens also obviates the need for two-tier testing. Preliminary results from our Phase I study also suggest that our assay has improved sensitivity for detecting early infections, particularly when the current assays fail.